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Abstract

One of the dose‑limiting toxicities of irinotecan hydrochloride (CPT‑11) is delayed‑onset diarrhea. CPT‑11 is converted to its active metabolite, SN‑38, which is conjugated to SN‑38 glucuronide (SN‑38G). SN‑38G excreted in the intestinal lumen is extensively deconjugated by bacterial β‑glucuronidase, resulting in the regeneration of SN‑38, which causes diarrhea. However, the deconjugation of SN‑38G by the intestinal microflora remains to be clarified. This study aimed to investigate the microbial transformation of SN‑38G by an anaerobic mixed culture of rat cecal microorganisms. Concentrations of SN-38G and SN-38 were then determined using high-performance liquid chromatography. Complete deconjugation of SN‑38G to SN‑38 in the mixed cultures was observed within 1 h of incubation, with 62.7% of the added SN‑38G being found in the supernatant. Approximately 80.4% of the SN‑38 in the supernatant was bound to protein, and the remaining 19.6% was detected as active free SN‑38. In total, only 12.3% (19.6 x 62.7%) of the SN‑38G added to the test tube was found in the supernatant in the ultrafiltrable free form, indicating that approximately 90% of the SN‑38G added to the growth medium either remained adsorbed onto the pelleted fraction or occurred in a protein‑bound form in the supernatant. The remaining 10% of the SN‑38G added to the growth medium existed in the unbound form, the form capable of causing damage to the intestinal membrane. In conclusion, these results indicated that the greater part of the SN‑38 produced from SN‑38G by the action of bacterial β‑glucuronidase is rapidly adsorbed onto intestinal bacterial cell walls or dietary fibers in pelleted fraction, and only 10% remains in the ultrafiltrable unbound form in the intestinal luminal fluid.

Introduction

Irinotecan hydrochloride (CPT-11) is a water-soluble
camptothecin derivative that inhibits topoisomerase I by
stabilizing the enzyme-DNA ‘cleavable complex’, causing
double-stranded DNA breaks during DNA replication and, ultimately,
cell death (1). CPT-11,
administered as a single agent or in combination with other drugs,
exhibited significant antitumor activity against various human
malignancies, including colorectal, small and non-small cell lung,
gastric, cervical and ovarian cancers (2). CPT-11 occasionally causes
unpredictable and severe neutropenia and/or diarrhea (3). Late-onset diarrhea remains one of the
as-yet unresolved problems linked to irinotecan administration and
has the potential to cause life-threatening dehydration and
electrolyte imbalance, which may necessitate premature
discontinuation of chemotherapy (2,4). The
current standard strategy for the treatment of CPT-11-induced
delayed-onset diarrhea is administration of high-dose loperamide
(5). Other therapeutic measures
that have been attempted include i) a Japanese herbal remedy (Kampo
medicine) (6) or antibiotics
(7–9) that inhibit intestinal β-glucuronidase
(7–9); ii) prevention of reabsorption of SN-38
and CPT-11 by oral alkalization (10) or administration of adsorbents,
activated charcoal (11) or AST-120
(12); and iii) various other
treatments, including encephalinase inhibitor (13), glutamine (14) and budesonide (15). However, the success of these
approaches has been limited.

The hydrophobic active metabolite, SN-38, is
generated from CPT-11 by carboxylesterase and then conjugated to
yield SN-38 glucuronide (SN-38G) by hepatic uridine diphosphate
glucuronosyl transferases (UGTs). SN-38G is an inactive metabolite
and is secreted in the duodenum (16–18).
Deconjugated SN-38 exerts a toxic effect when it comes into contact
with the intestinal mucosa, and deconjugation of SN-38G to SN-38 by
β-glucuronidase in the intestinal microflora is considered to be a
major contributory factor in the development of CPT-11-induced
late-onset diarrhea (11,19–24).

To the best of our knowledge, however, as yet no
studies have been conducted on the biotransformation of SN-38G by
the intestinal microflora, even though this knowledge is essential
in achieving a precise understanding of the mechanism by which
CPT-11 induces late-onset diarrhea. The purpose of this study was
to investigate the microbial transformation of SN-38G by using an
anaerobic mixed culture of rat cecal microorganisms.

Animals

The study was approved by the Institutional Animal
Care and Use Committee of Yakult Central Institute. The animals
comprised 2 10-week-old male Wistar rats, weighing 271.6 g and
273.0 g. The animals were obtained from Japan SLC (Hamamatsu,
Japan) and reared in cages with a wire-mesh bottom in a
temperature- and humidity-controlled animal facility under a 12-h
light-dark cycle. The animals received a steam-sterilized
commercial diet, and had free access to food and water during the
acclimatization period.

Cecal contents

The rats were sacrificed by exsanguination under
pentobarbital anesthesia. The cecum was removed and the contents
collected and transferred within 5 min to an anaerobic porter
(Terumo, Tokyo, Japan). Cecal contents (4 g) from the two rats were
diluted approximately 10-fold in 40 ml sterile, pre-reduced
peptone-yeast extract (PY) broth (see below) under flushing with
N2.

Culture experiment

The basal medium used in this experiment was PY
broth, which consisted of 1 g polypeptone (Daigo-Eiyo Chemical Co.,
Tokyo, Japan); 1 g yeast extract (Difco Laboratories, Detroit, MI,
USA); 4 ml salt solution recommended by Holdeman et al
(25); and 0.05 g l-cysteine-HCl
per 100 ml in 0.02 M phosphate buffer, pH 7.5, sterilized at 120˚C
for 15 min. A 1:10 suspension of the cecal contents in the
peptone-yeast extract broth was incubated for a specified period of
time under an atmosphere of pure N2 after the addition
of SN-38G. The culture medium was then analyzed. Assays were run in
triplicate for samples incubated with SN-38G and the data expressed
as the mean ± standard deviation.

SN-38G-containing medium

SN-38G was dissolved in methanol at 1 mg/ml. After
sterilization by passage through a 0.45-μm membrane filter, 0.3 ml
SN-38G solution was added to 30 ml cecal content suspension (1:10).
The medium was then dispensed in 1.5-ml aliquots into 15×105-mm
test tubes.

Incubation

For anaerobic growth, the tubes containing SN-38G
were inoculated under flushing with N2, tightly
stoppered and incubated for 0, 1, 3, 6, 24, 48, or 72 h at 37˚C.
The time-point immediately following inoculation was denoted as 0
h. The N2 gas was used after removal of any residual
oxygen by passing it over heated copper gauze.

Extraction and high-performance liquid
chromatographic analysis of SN-38G and SN-38

The inoculated tubes were assayed for SN-38G or its
metabolite, SN-38, at various time-points during incubation under
N2 for up to 72 h. Spent culture medium (1 ml) was
centrifuged at 15,000 rpm for 1 min at 0˚C and the supernatant was
collected. For the assay of the whole culture medium or its
supernatant, 0.1 ml spent culture medium or the supernatant
described above was mixed with 0.4 ml methanol and centrifuged at
15,000 rpm for 1 min at −10˚C. Each supernatant (50 μl) was diluted
with 0.2 ml of 0.15 M H3PO4 and 0.25 ml
internal standard solution containing 1 μg/ml camptothecin. To
separate non-protein-bound compounds from protein-bound compounds,
supernatant ultrafiltrates were obtained by centrifugation at 2,000
g and 4˚C for 20 min in the ultra-free®-MC 30,000 NMWL
filter unit (Centriplus Millipore Corporation, Bedford, MA, USA),
and were then processed by the same means as described for the
spent culture medium and supernatant. Each sample was fresh-frozen
at −20˚C until analysis.

Concentrations of SN-38G and SN-38 were determined
using a high-performance liquid chromatographic (HPLC) method with
a fully automated on-line solid phase extraction system (Spark
Holland, Emmen, The Netherlands) as previously described (26). The quantification limit for SN-38G
and SN-38 was 100 ng/ml and 10 ng/ml, respectively.

For analysis of the concentrations of the lactone
and carboxylate forms of SN-38, 0.3 ml culture medium or its
supernatant was mixed for 5 sec with 0.3 ml methanol, which was
previously chilled in a dry, ice-cold isopropanol bath and
immediately centrifuged at 15,000 rpm for 1 min at −10˚C. The
obtained supernatant was rapidly poured into a vial, set on an
autosampler at 4˚C and analyzed by the HPLC system. The
concentrations of the SN-38 lactone and carboxylate forms were
determined using HPLC according to the method reported by Kaneda
et al (27). The lower
limits of quantification for the SN-38 lactone and carboxylate
forms were 1 ng/ml and 10 ng/ml, respectively.

The percentages of the protein-bound compounds
(SN-38 and SN-38G) were determined as follows: i) concentration of
protein-unbound compounds in supernatant = A × B/C, where A is the
concentration of the compound in ultrafiltrate, B is the weight of
the ultrafiltrate after centrifugation and C is the weight of the
supernatant applied to the ultrafilter unit; ii) percentage of
protein-bound compounds (%) = (A - B)/A × 100, where A is the
concentration of the compounds in supernatant and B is the
concentration of the protein-unbound compounds in supernatant.

Results

Deconjugation of SN-38G in the culture of
cecal content

Cecal content (diluted 10-fold) was grown
anaerobically in PY broth in the presence of SN-38G. Immediately
after the start of incubation, the percentage of SN-38G in the
supernatant was found to be 46.9%, while in the pelleted fraction
it was 13.8% (Fig. 1). The
remaining 39.4% was identified as deconjugated SN-38 (33.3% in the
supernatant and 6.1% in the pelleted fraction), reflecting the
rapid deconjugating activity of the cecal microflora. At 1 h after
incubation, most of the SN-38G was converted to SN-38 by bacterial
β-glucuronidase, and little SN-38G was subsequently detectable in
the culture medium.

Change in percentage concentration of
lactone and carboxylate forms of SN-38 during the study period

Immediately following the addition of SN-38G to the
culture medium, the metabolites (deconjugated SN-38) were in the
lactone form, irrespective of whether they were derived from the
pelleted fraction or the supernatant (Fig. 1). At 1 h after the start of
incubation, 54.4 and 37.0% of the total SN-38 was found in the
lactone form, and 8.3 and 0.3% in the carboxylate form in the
pelleted fraction and supernatant, respectively. From 3 up to 72 h
of incubation, the lactone to carboxylate ratio remained relatively
constant at 9:1. In the supernatant, the lactone-to-carboxylate
ratio was 85:15, with this ratio remaining stable up to 72 h
(Fig. 1).

Change in the percentage composition of
SN-38 in the pelleted fraction and supernatant during the study
period

Immediately following the addition of SN-38G, 19.9%
was found in the pelleted fraction (13.8% as SN-38G and 6.1% as
SN-38 lactone) (Fig. 1). After 1 h
of incubation, 37.0 and 0.3% in the pelleted fraction, and 54.4 and
8.3% in the supernatant was found in the lactone and carboxylate
forms of SN-38, respectively. The percentage of the lactone form of
SN-38 in the pelleted fraction gradually increased from 40.8% after
3 h to 52.8% after 72 h of incubation. The bulk of the SN-38 in the
pelleted fraction was in the lactone form, which may be the main
form adsorbed onto bacterial cells in the pelleted fraction due to
its lipophilic nature. Almost no metabolites were detected in the
carboxylate form in the pelleted fraction.

Binding of SN-38G and SN-38 to protein in
cell-free supernatant

The percentages of protein-bound SN-38G and SN-38 in
the cell-free supernatant are shown in Table I. Immediately after the start of
incubation, 81.3% of SN-38G and 54.1% of SN-38 lactone in the
supernatant was bound to protein. At 1 h after the start of
incubation, 80.4% of SN-38 in the supernatant (SN-38 lactone, 54.4%
and SN-38 carboxylate, 8.3%) was bound to protein. At 3 h after the
start of incubation, the bulk of the SN-38 was bound to protein,
and the percentage of the protein-bound form was relatively
constant (85.8 to 91.7%). The congregated lactone gradually moved
from the supernatant to the pelleted fraction, with the percentage
of the lactone form in the supernatant gradually decreasing from
54.4% after 1 h to 37.8% after 72 h of incubation. This decrease
may be attributable to a transition from the aqueous solution to
the pelleted fraction due to precipitation of the protein-bound
lactone.

Table I

Percentages of protein-bound SN-38G
and SN-38 in the cell-free supernatanta.

Table I

Percentages of protein-bound SN-38G
and SN-38 in the cell-free supernatanta.

Incubation time
(h)

Composition (%)

Protein-bound
compound in supernatant

Rate of
protein-unbound compound in supernatant to total SN-38G added
(%)b

a Change in composition in the sediment and
supernatant and protein-binding of SN-38 and SN-38 glucuronide
(SN-38G) when SN-38G was added at 10 μg/ml to mixed cecal content
culture carried out under anaerobic conditions.

c At 1 h after incubation, the majority of SN-38G
was converted to SN-38 by bacterial β-glucuronidase, and little
SN-38G was subsequently detected in the culture medium. Data are
expressed as the mean ± SD of the experiment performed in
triplicate.

Discussion

We have previously shown that little SN-38 was found
in the supernatant when SN-38 lactone was added to growth medium,
and that a significant quantity was rapidly adsorbed by intestinal
bacterial cell walls in the pelleted fraction (28). In this experiment, extensive
deconjugation of SN-38G to SN-38 occurred in mixed cecal cultures
carried out under anaerobic growth conditions, as reported by
Takasuna et al (22). As a
result, almost all of the SN-38 metabolites found in the test tubes
were deconjugated after 1 h of incubation (Fig. 1). The cytotoxic mechanism of SN-38
was originally ascribed to the closed lactone form, and opening of
the lactone ring (carboxylate form) results in the loss of
anticancer activity (29). In the
present study, at 1 h after the start of incubation, 91.4% was
found in the active lactone form of SN-38 (37.0% in the pelleted
fraction and 54.4% in the supernatant) and 8.6% in the carboxylate
form; in other words, 62.7% of the added SN-38G was found in the
supernatant (Table I). Furthermore,
80.4% of the SN-38 in the supernatant was bound to protein, and
19.6% was detected as the active free form of SN-38. In total, only
12.3% (19.6 × 62.7%) of SN-38G added to the test tube was found in
the supernatant as the ultrafiltrable, free non-protein-bound form.
The percentage of free SN-38 gradually decreased from 12.3% after 1
h to 6.4% after 72 h of incubation. This decrease indicates that
more than 90% of the SN-38G added to the growth medium was either
adsorbed onto the bacterial cell membrane in the pelleted fraction
or dietary fibers, or existed in the protein-bound form in the
supernatant after 1 h of incubation. It is noteworthy that only
approximately 10% of the SN-38G added to the growth medium existed
in the free unbound form.

Deconjugation of SN-38G to SN-38 by β-glucuronidase
in the intestinal microflora is considered to be a major
contributory factor in the intestinal toxicity of CPT-11, which is
believed to result from contact of this deconjugated SN-38 with the
intestinal mucosa (11,19–24).
Unlike other camptothecin drugs, the SN-38 lactone form
preferentially binds to blood proteins as compared with the
corresponding carboxylate form, resulting in significant stability
in the presence of human plasma (30). Furthermore, since the lactone form
of SN-38 is highly water-insoluble, SN-38 is rapidly and tightly
bound to hydrophobic bacterial cell walls or dietary fibers
(Fig. 1) (28), which may explain the reason that
little free SN-38 remained in the lactone form in the supernatant
of the 10-fold diluted culture in this study. This event
potentially reduces the availability of the active metabolite,
SN-38, to the intestinal mucosa.

Hidaka et al (12) studied the adsorption of CPT-11 onto
oral adsorbent AST-120 (Kremezin) for the prevention of
delayed-onset diarrhea. It was presumed that the camptothecin drug
may not have been completely adsorbed onto the Kremezin, since it
failed to completely prevent diarrhea. A preliminary study by
Takasuna et al (31) did not
confirm the anti-diarrheal activity of D-glutaric acid-1,4-lactone
monohydrolate, a specific β-glucuronidase inhibitor. However, the
dose of D-glutaric acid-1,4-lactone was sufficient to inhibit
β-glucuronidase. CPT-11-induced delayed-onset diarrhea is presumed
to be attributable to damage to the small intestine, rather than
the cecum, where β-glucuronidase activity in the lumen is highest.
In their study, Fittkau et al (32) reported that D-saccharic acid
1.4-lactone alleviated CPT-11-induced mucosal damage in the small
intestine, the luminal contents of which almost completely lack
β-glucuronidase (22). Therefore,
these authors suggested that a mechanism other than β-glucuronidase
inhibition in the intestinal lumen may be involved (31). Furthermore, Kurita et al
(33) carried out a CPT-11-induced
diarrhea study using Gunn rats, which have an inherent deficiency
of uridine diphosphate glucuronosyl transferase 1A and cannot
conjugate SN-38 to SN-38G. The onset of CPT-11-induced diarrhea in
Gunn rats was not affected by β-glucuronidase activity. The
alleviation of diarrhea by streptomycin in the Gunn rats indicated
that streptomycin exerted its effect by a mechanism other than the
inhibition of β-glucuronidase.

In conclusion, the results of the present study have
shown that the extensive conversion of SN-38G to SN-38 occurred in
mixed cecal content cultures grown under anaerobic growth
conditions. Due to the highly hydrophobic nature of SN-38, a
significant quantity of SN-38 was adsorbed onto the intestinal
microflora and dietary fibers in the pelleted fraction, or became
bound to protein in the cell-free supernatant. Additionally, only
10% of SN-38 was detected as the protein-unbound form in the
supernatant in vitro. Further studies are therefore required
to identify the exact role of the intestinal microflora in
CPT-11-induced late-onset diarrhea.